Cellulose in General (Plant)
Cellulose is the most abundant biological macromolecule on the planet earth. It forms the basic structural matrix of the cell walls of nearly all plants, many fungi and some algae. It is a major biopolymer of tremendous economic importance as it find multifarious uses in industries such as textiles, pulp and paper, cosmetics, healthcare, food, audio products, sport goods, etc, as well as in the preparation of cellulose derivatives such as cellophane, rayon, cellulose acetate and few others. Apart from this, cellulose is also used for environmental remediation especially in treating oil spills and removing toxic materials. The demand for cellulose has traditionally been met by wood and cotton, which contain over 50% and over 94% cellulose, respectively. However, plant resources cannot sustain an increasing demand for cellulose requirements due to fast diminishing forest resources, decreased land holdings for agriculture and other environmental concerns. This along with the difficulty in removal of hemicellulose or lignin inherently associated with cellulose limits its applications. It, therefore, necessitates a search for a commercially viable alternative to plant cellulose.
Microbial Cellulose as an Alternative to Plant Cellulose and its Importance
Microbial cellulose has emerged as an important and viable alternative to plant cellulose. Since ages, cellulose is recognized as the major component of plant biomass. However, it also represents a major chunk of microbial extracellular polymers. The cellulose produced by microbes is called microbial cellulose (MC). It is an exopolysaccharide. Some bacteria are in condition to produce cellulose, as reported from the strains of the genera Gluconacetobacter (formerly Acetobacter), Agrobacterium, Pseudomonas, Rhizobium and Sarcina. 
The production of cellulose by Acetobacter xylinum was reported for the first time in 1886 by A. J. Brown. He observed that the resting cells of Acetobacter produced cellulose in the presence of oxygen and glucose. This non-photosynthetic organism can procure glucose, glycerol, or other organic substrates and can convert them into pure cellulose. A. xylinum was reported as the most efficient producer of MC. The production of cellulose can be carried out in either solid-phase cultivation or submerged culture. Investigations have been focused on the mechanism of biopolymer synthesis, as well as on its structure and properties, which determine practical use thereof (Legge, 1990; Ross et al., 1991). Acetobacter xylinum produces two forms of cellulose: Cellulose I, the ribbon like polymer, and Cellulose II, the thermodynamically more stable amorphous polymer. Plant and bacterial cellulose are chemically the same, β 1, 4 glucans, having same molecular formula (C6H10O6)n but their physical features are different (Yoshinaga et al., 1997). As compared to plant cellulose, bacterial cellulose is chemically purer, has a high degree of crystallinity, polymerization, tensile strength, shear resistance and high water holding capacity. Fibrils of bacterial cellulose are about 100 times thinner than that of plant cellulose, making it more porous material.
Research on microbial cellulose production has been sporadically attempted with an increased impetus after 1990s. Production of microbial cellulose was carried out either in static or shaking culture. Glucose is supposed to be the most common carbon source for microbial cellulose production. However, there are many reports of microbial cellulose production using other carbon sources. In a study by Oikawa et al (1995) microbial cellulose production is carried out using D-Arabitol by Acetobacter xylinum. Similarly, Yang et al (1998) carried out the production of microbial cellulose by Acetobacter xylinum under shaking conditions using glucose, fructose, and sucrose individually and in combination. Microbial cellulose production by Acetobacter xylinum has been also attempted using D-xylose as a carbon source by Ishihara et al (2002). Keshk and Sameshima (2005) evaluated the effect of different carbon sources on the production of bacterial cellulose by Acetobacter xylinum and found that glycerol gave the highest yield of bacterial cellulose. In 2008, Hong and Qiu, developed a new carbon source from konjac powder that enhanced production of bacterial cellulose by A. aceti subsp. xylinus in static cultures.
Optimization studies on the production of microbial cellulose can enhance the yield both in static and shaking condition. Many scientists have attempted to optimize the culture conditions in order to enhance microbial cellulose production. In 2002, Heo and Son developed an optimized, simple and chemically defined medium for bacterial cellulose production by Acetobacter sp. V9 in shaking culture. In 2005, Bae and Shoda, statistically optimized the culture conditions for the bacterial cellulose production in shaking condition by Acetobacter xylinum using response surface methodology. Kim et al (2006) developed an optimized medium for the production of microbial cellulose in static condition by Glucanocetobacter sp. isolated from persimmon vinegar. An optimized medium for microbial cellulose production in static condition by Acetobacter sp. 4 B-2 was developed by Pourramezan et al., (2009), who studied the bacterial cellulose production using two categories of carbon sources (monosaccharides and disaccharides) and found sucrose to be the best carbon source for cellulose production. Jung et al., (2010) used a cost effective molasses-corn steep liquor medium for microbial cellulose production under shaking culture conditions by Acetobacter sp. V6.
Microbial cellulose yield in static cultures is mostly dependent on the surface/volume ratio. Microbial cellulose synthesis in static conditions can be achieved either in a one step [as attempted by most of the workers] or a two-step procedure using agitated fermentation followed by the static culture (Okiyama et al., 1992). They also scaled up the production upto 800 ml.
Attempts to produce microbial cellulose using conventional fermentors in order to scale up production in agitated condition have yielded few significant results. Bungay and Serafica (U.S. Pat. No. 6,071,727, 2000) worked on the production of microbial cellulose using a rotating disc or linear conveyer bioreactor. Chao et al (2000) used an airlift reactor for the production of microbial cellulose by Acetobacter xylinum. Tung et al (1997) modified the airlift reactor to improve the performance of fermentation processes. The production of microbial cellulose by Acetobacter xylinum was carried out in a jar fermentor and the effect of the pH and dissolved oxygen on production was observed (Hwang et al., 1999). In 2005, Bae and Shoda produced bacterial cellulose by Acetobacter xylinum subsp. sucrofermentans using molasses medium in a jar fermentor.
Microbial cellulose can be dried either by freeze drying, air drying, vacuum oven drying or drying in a simple oven. Most of the workers have dried microbial cellulose in a vacuum oven (Chao et al., 2000, Bae and Shoda, 2005, Kim et al., 2006 and Pourramezan et al., 2009). The purified bacterial cellulose pellets were dried to a constant weight at 80 to 105 degree C. in a conventional oven (Hwang et al., 1999 and Son et al., 2001). Harris et al., 2010 (U.S. Pat. No. 7,709,631 B2), have air dried the microbial cellulose mats at 37° C. using polypropylene mesh as base for drying.
Reference may be made to the study of Kim et al (2006) which utilizes a bacterial strain Gluconacetobacter sp. RKY5 for cellulose production. The strain was isolated from persimmon vinegar as opposed to the strain of Gluconacetobacter oboediens isolated in the present invention from fruits residue. Also, the bacterial strain Gluconacetobacter oboediens is novel cellulose producing bacterial strain and has not been yet reported to produce cellulose. This is the first report of cellulose production by Gluconacetobacter oboediens and that too with much higher yield. The higher microbial cellulose yield by the bacterium of the present invention can be explained on the fact that Gluconacetobacter oboediens MTCC 5610 is more potent than the strain of Kim et al (2006). Also, the difference in the final yields of microbial cellulose in the present study and the study of Kim et al lies in the method of process optimization. After process optimization w.r.t. different parameters by “one variable at time approach” and then, “Response Surface Methodology”, (statistical optimization), the inventors of the present invention achieved a maximum microbial cellulose production of 11.8 g/L, which can be further increased by auxiliary optimization experiments. However, it may be noted that Kim et al (2006), have optimized the process parameters by only “one variable at a time approach” and no statistical optimization was carried out in their study. Further, the process of the present invention is more economic and simple for microbial cellulose production as compared to their process.
Consequently, by all the facts reported above it can be concluded that the bacterial strain Gluconacetobacter oboediens MTCC 5610 is different from the strains reported in the prior art for microbial cellulose production. Further, the process optimization for achieving higher yields was more efficient and economic than that carried out by the earlier studies.
In summary, the drawbacks of the hitherto reported literature can be summarized as follows:                There are only few reports on the microbial cellulose production by newer species of Acetobacter (Gluconacetobacter) and other bacterial strains. Most of the work on microbial cellulose has been carried out using Acetobacter xylinum, which is the most common and well known cellulose producer.        Most of the researches have been conducted only upto flask level, (i.e 30 or 50 ml production medium in a 250 ml Erlenmeyer flask) and a calculated yield per liter is presented. These results do not clearly explain the scalability of the production.        There are few reports of microbial cellulose production in static culture condition providing significant titers. There is no report directly related to the scale up of microbial cellulose production in static culture. Most of the workers have scaled up the production in agitated culture either in a jar fermentor or airlift fermentor. Static culture is important as it produces microbial cellulose in a sheet or mat form which is essential for some important applications of microbial cellulose especially in the medical field as wound dressings, artificial skin substitute, material for arterial implants and others.        Detailed description on the drying method and recovery of the microbial cellulose therefrom has not been presented by any of the workers. Drying step is very important as it gives the final dry weight i.e final yield of the microbial cellulose produced. There is only one patent by Harris et al., 2010 (U.S. Pat. No. 7,709,631 B2) which has explained the air drying process of microbial cellulose, wherein they have kept the microbial cellulose mats between two pieces of polypropylene mesh and further, kept them in an incubator at 37° C. for 18-36 h. However, the use of polypropylene mesh and incubator thereby for drying is less economical as compared to the process used in the present invention i.e. drying on a wooden plank and a porous fabric which is quite economical.        